The long-term goal of our work is to understand the molecular mechanisms that control eukaryotic mRNA stability, which is a critical step in gene regulation. One major pathway of mRNA turnover in eukaryotes initiates with poly(A) tail shortening, which triggers decapping, leading to 5' to 3' exonucleolysis. Decapping is a key step in this pathway because it induces degradation of the mRNA, and is the site of numerous control inputs including the poly(A) tail and specific sequences that modulate mRNA decapping rate. Given this importance, we focused on understanding the mechanisms of mRNA decapping. In the past funding period, we clarified the nature of the decapping enzyme, identified the mechanisms by which some proteins affect the rate of decapping, and provided strong evidence that a key step in decapping is exiting the translating pool of mRNAs coupled with a specific mRNP re-arrangement. In addition, we also identified novel cytoplasmic structures (referred to as P-bodies) where the mRNA decapping machinery is concentrated and where mRNA degradation can occur. P-bodies are significant because: 1) they imply unanticipated compartmentalization of mRNA decay, which becomes a possible point of mRNA regulation, 2) are conserved in other eukaryotes and 3) are likely to be sites of additional events in mRNA biology, possibly including storage of translationally repressed mRNAs. In this grant, we will continue our analysis of mRNA decapping with a focus on understanding the composition, properties, and function of P-bodies. The specific aims are as follows: I) Determine the composition and ultrastructure of P-bodies. II) Analyze P-body assembly. III) Analyze the function of the Dhh1 and Pat1 proteins. IV) Determine if P-bodies function in other aspects of cytoplasmic mRNA physiology.